RESUMEN
The assortment of cellular microRNAs ("microRNAome") is a vital readout of cellular homeostasis, but the mechanisms that regulate the microRNAome are poorly understood. The microRNAome of glioblastoma is substantially down-regulated in comparison to the normal brain. Here, we find malfunction of the posttranscriptional maturation of the glioblastoma microRNAome and link it to aberrant nuclear localization of DICER, the major enzymatic complex responsible for microRNA maturation. Analysis of DICER's nuclear interactome reveals the presence of an RNA binding protein, RBM3, and of a circular RNA, circ2082, within the complex. Targeting of this complex by knockdown of circ2082 results in the restoration of cytosolic localization of DICER and widespread derepression of the microRNAome, leading to transcriptome-wide rearrangements that mitigate the tumorigenicity of glioblastoma cells in vitro and in vivo with correlation to favorable outcomes in patients with glioblastoma. These findings uncover the mechanistic foundation of microRNAome deregulation in malignant cells.
Asunto(s)
Glioblastoma , MicroARNs , Glioblastoma/genética , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular , Proteínas de Unión al ARN/genéticaRESUMEN
Each year, 1 million children die due to perinatal asphyxia; however, there are no effective drugs to protect the neonatal brain against hypoxic/ischemic damage. In this study, we demonstrated for the first time the neuroprotective capacity of 3,3'-diindolylmethane (DIM) in an in vivo model of rat perinatal asphyxia, which has translational value and corresponds to hypoxic/ischemic episodes in human newborns. Posttreatment with DIM restored the weight of the ipsilateral hemisphere and normalized cell number in the brain structures of rats exposed to perinatal asphyxia. DIM also downregulated the mRNA expression of HIF1A-regulated Bnip3 and Hif1a which is a hypoxic marker, and the expression of miR-181b which is an indicator of perinatal asphyxia. In addition, DIM inhibited apoptosis and oxidative stress accompanying perinatal asphyxia through: downregulation of FAS, CASP-3, CAPN1, GPx3 and SOD-1, attenuation of caspase-9 activity, and upregulation of anti-apoptotic Bcl2 mRNA. The protective effects of DIM were accompanied by the inhibition of the AhR and NMDA signaling pathways, as indicated by the reduced expression levels of AhR, ARNT, CYP1A1, GluN1 and GluN2B, which was correlated with enhanced global DNA methylation and the methylation of the Ahr and Grin2b genes. Because our study provided evidence that in rat brain undergoing perinatal asphyxia, DIM predominantly targets AhR and NMDA, we postulate that compounds that possess the ability to inhibit their signaling are promising therapeutic tools to prevent stroke.
Asunto(s)
Apoptosis/genética , Asfixia/tratamiento farmacológico , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Receptores de Hidrocarburo de Aril/genética , Receptores de N-Metil-D-Aspartato/genética , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Asfixia/genética , Asfixia/patología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Metilación de ADN/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Indoles/farmacología , Recién Nacido , Masculino , Proteínas de la Membrana/genética , MicroARNs/genética , Proteínas Mitocondriales/genética , N-Metilaspartato/farmacología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , RatasRESUMEN
Training chicks (Gallus domesticus) on a one-trial passive avoidance task results in transient and time-dependent enhanced increases in N-methyl-d-aspartate- or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-stimulated intracellular calcium concentration in synaptoneurosomes isolated from a specific forebrain region, the intermediate medial hyperstriatum ventrale. This increase could result from either calcium entry from the extracellular medium or from mobilization of intracellular calcium stores. We have therefore examined the effects of dantrolene, an inhibitor of calcium release from the intracellular ryanodine-sensitive store, on these processes. Dantrolene, 50 nmol per hemisphere injected intracerebrally 30 min pre- or 30 min posttraining, blocked longer term memory for the passive avoidance task, whereas memory for the task was unaffected when dantrolene was injected at earlier or later times. Preincubation of synaptoneurosomes, isolated from the intermediate hyperstriatum ventrale 10 min after training, with 100 nM dantrolene abolished the enhanced training-induced increase in intracellular calcium concentration elicited by 0.5 mM N-methyl-d-aspartate. By contrast, the training-induced enhancement of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-stimulated increase in intracellular calcium concentration in synaptoneurosomes prepared 6 h posttraining was unaffected by preincubation with dantrolene, which was not amnestic at this time. Calcium release from ryanodine-sensitive intracellular stores may thus be a necessary stage in the early phase of the molecular cascade leading to the synaptic modulation required for long-term memory storage.
Asunto(s)
Calcio/metabolismo , Insecticidas/farmacología , Memoria/efectos de los fármacos , Rianodina/farmacología , Animales , Reacción de Prevención/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Pollos , Dantroleno/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Transporte Iónico/efectos de los fármacos , Relajantes Musculares Centrales/farmacología , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacología , Prosencéfalo/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
This in vivo study, aimed at detecting the N-methyl-D-aspartate (NMDA) evoked Ca(2+)-induced Ca(2+) release from intracellular stores in the neonatal rat brain, demonstrates that the application of 5 mM N-methyl-D-aspartate via a microdialysis probe for 20 min to the dentate gyrus (DG) of halotane-anesthetized 7 day-old (postnatal day 7, PND 7) rats induces a prolonged decrease in Ca(2+) concentration in an initially calcium-free dialysis medium, indicative of a drop in the extracellular concentration of Ca(2+) and Ca(2+) influx to neurons. In parallel experiments, a huge NMDA-evoked release of 45Ca from the pre-labeled endogenous Ca(2+) pool was observed and interpreted as the expression of intracellular Ca(2+) release. Dantrolene (100 microM) significantly inhibited the NMDA-induced 45Ca release, whereas 250 microM ryanodine exerted an unspecific biphasic effect. Autoradiographic and immunocytochemical detection of ryanodine receptors and calbindin D(28K), respectively, in the hippocampal region of PND 7 rats displayed a pronounced expression of [3H]ryanodine binding sites in the DG, but only a slight immunoreactivity of calbindin D(28K). Plastic changes in neurons or excitotoxic neuronal damage induced by the activation of NMDA receptors are mediated by Ca(2+) signals, resulting from an influx of extracellular Ca(2+), and also in some neurons, from the release of intracellular Ca(2+). Our previous in vivo microdialysis experiments visualized NMDA-evoked 45Ca release in the adult rat dentate gyrus, attributable to Ca(2+)-induced Ca(2+) release from the ryanodine-sensitive pool. An additional role of calbindin in the mechanism of this phenomenon has been suggested. This aspect has not been studied in vivo in newborn rats. Our present results indicate that the release of 45Ca from the prelabeled intracellular, dantrolene-sensitive Ca(2+) pool in the DG neurons of immature rats, most probably representing a phenomenon of Ca(2+)-induced Ca(2+) release, significantly participates in the generation of NMDA receptor-mediated intracellular Ca(2+) signals, whereas the role of calbindin D(28K) in the mechanism of 45Ca release is negligible.
Asunto(s)
Animales Recién Nacidos/metabolismo , Calcio/metabolismo , Giro Dentado/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , N-Metilaspartato/farmacología , Animales , Autorradiografía , Calbindinas , Radioisótopos de Calcio , Dantroleno/farmacología , Giro Dentado/efectos de los fármacos , Inmunohistoquímica , Microdiálisis , Relajantes Musculares Centrales/farmacología , Ratas , Ratas Wistar , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteína G de Unión al Calcio S100/metabolismoRESUMEN
In vivo microdialysis combined with the measurement of (45)Ca(2+) efflux from prelabelled hippocampus demonstrated a pronounced N-methyl-D-aspartate (NMDA)-evoked (45)Ca(2+) release to the dialysate in the rat dentate gyrus (DG) and CA1, whereas in rabbit a slight release of (45)Ca(2+) was observed only in the DG. In vitro, we noticed that the NMDA-evoked increase in Fura-2 detected intracellular Ca(2+) concentration in synaptoneurosomes from the rat, but not from the rabbit hippocampus, was strongly inhibited by the ryanodine receptor (RyR) antagonists dantrolene and ryanodine. To establish the mechanism of these differences, we characterised their possible dependence on the expression of RyR and their co-localisation with the calcium binding protein calbindin D(28k). A pronounced expression of [(3)H]ryanodine binding sites in the rat DG, which is only slight in the CA1, was demonstrated whereas in rabbit they were only found in the DG. The pattern of expression of calbindin D(28k) immunoreactivity and RyR in the rat and rabbit hippocampus was similar. These results suggest that the functional role of RyR in the generation of the NMDA receptor-mediated intracellular Ca(2+) signalling in the rabbit hippocampal neurones is marginal when compared to the rat. These differences reflect a diverse expression of RyR in both species. The corresponding differences in calbindin D(28k) immunoreactivity are most probably secondary in nature.
Asunto(s)
Señalización del Calcio/fisiología , Hipocampo/metabolismo , Neuronas/metabolismo , Conejos/metabolismo , Ratas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Calbindinas , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Masculino , Microdiálisis , N-Metilaspartato/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Ensayo de Unión Radioligante , Ratas Wistar/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , TritioRESUMEN
The mitochondrial permeability transition (MPT) resulting from calcium-induced opening of cyclosporin A (CsA)-sensitive megachannels, leading to deenergisation of mitochondria and release of pro-apoptotic cytochrome c, has been implicated in the pathomechanism of excitotoxic neurodegeneration. The aim of this work was to test neuroprotective potential of CsA in the model of N-methyl-D-aspartate-(NMDA)-induced excitotoxicity in vivo, and to verify utility of microdialysis of the rabbit hippocampus in vivo for these mechanistic studies. In vitro experiments demonstrated that the early rapid phase of Ca(2+)-induced swelling of isolated brain mitochondria, and of accompanying cytochrome c release, was strongly inhibited by 0.5 microM CsA. In the in vivo experiments 1 mM NMDA was applied for 20 min to the hippocampus in a control, or 5 microM CsA-containing dialysis medium via transhippocampal microdialysis probes, and changes in extracellular Ca2+ concentration and in NO release were monitored. Application of NMDA induced a prolonged decrease in the extracellular concentration of Ca2+, reflecting influx of Ca2+ to stimulated neurones. CsA only slightly enhanced this effect. NMDA induced also release of NO to the dialysis medium. Morphological examination 30 min after NMDA application visualised swelling of dendritic mitochondria and cisternae of endoplasmatic reticulum of pyramidal neurones in the CA1 sector of the hippocampus in the vicinity of microdialysis probes. CsA prevented mitochondrial swelling. After 24 h degeneration of the CA1 pyramidal neurones close to a microdialysis probes was observed, which was partially prevented in CsA-treated rabbits. These results indicate that the mechanism of CsA nuroprotection may be at least in part ascribed to prevention of MPT. Microdialysis of the rabbit hippocampus combined with NMDA excitotoxicity appeared to be useful in mechanistic studies of CsA neuroprotection.
Asunto(s)
Ciclosporina/farmacología , Hipocampo/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Hipocampo/patología , Microdiálisis , N-Metilaspartato/toxicidad , Óxido Nítrico/biosíntesis , Conejos , Ratas , Ratas WistarRESUMEN
The aim of this in vivo microdialysis study was to characterise the regulation of prostaglandin D2 (PgD2) synthesis by NMDA receptors in the rabbit hippocampus in relation to changes in extracellular Ca2+ concentration ([Ca2+]e) and nitric oxide (NO) levels. Samples of dialysate were analysed for changes in PgD2 concentration, in [Ca2+]e and in the level of NO. The results demonstrated that a 20-min pulse application of 0.1-2.5 mM NMDA via a microdialysis probe induced a prolonged stimulation of PgD2 release that was sensitive to competitive NMDA receptor antagonists. An inhibitor of voltage-sensitive Na+ channels, tetrodotoxin, did not influence this effect but significantly suppressed basal PgD2 production, whereas a NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), prevented NMDA-evoked NO release and inhibited NMDA-induced PgD2 release in an L-arginine-sensitive manner. NO donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, stimulated PgD2 release. NMDA-evoked decrease in [Ca2+]e was insensitive to TTX and L-NAME. These results demonstrate an in vivo NMDA receptor-mediated modulation of PgD2 synthesis in the brain, in which NO participates.
Asunto(s)
Calcio/fisiología , Hipocampo/fisiología , N-Metilaspartato/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/fisiología , Penicilamina/análogos & derivados , Prostaglandina D2/biosíntesis , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Espacio Extracelular/fisiología , Hipocampo/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Penicilamina/farmacología , Conejos , S-Nitroso-N-Acetilpenicilamina , Tetrodotoxina/farmacologíaRESUMEN
A temporal cascade of events has been described from a number of biochemical investigations of passive avoidance training in day-old chicks. Among these, within minutes of training, there is a transient, enhanced release of glutamate and increased agonist and antagonist binding to N-methyl-D-aspartate-sensitive glutamate receptors in the intermediate medial hyperstriatum ventrale of the forebrain. Some 6.5 h later, alpha-amino-3-hydroxy-5-methyl-4-isoxazo lepropionate binding to glutamate receptors is also increased in the same region. These processes might be predicted to affect the uptake of calcium via voltage-sensitive calcium channels or glutamate receptor-associated channels, thereby changing the intracellular calcium concentration. To test this possibility, we have measured the calcium concentration in synaptoneurosomes, containing both pre- and postsynaptic elements, prepared from left and right intermediate medial hyperstriatum ventrale at various times following training, using Fura 2-AM as the indicator of intracellular calcium concentration. Synaptoneurosomes, prepared immediately and 5 min after training, were stimulated with 70 mM potassium chloride in the presence of 2 mM calcium, resulting in a significantly enhanced increase in calcium concentration in synaptoneurosomes from the left hemisphere of trained chicks. This effect was absent in samples obtained at later times after training. N-Methyl-D-aspartate (0.5 mM) induced a significant enhancement in the increase in calcium concentration in intermediate medial hyperstriatum ventrale from both left and right hemispheres 10 min and 30 min after training. At 3 h and 6 h after training, alpha-amino-3-hydroxy-5-methyl-4-isoxazo lepropionate (0.5 mM) induced a significantly enhanced increase in calcium concentration in samples from either hemisphere. These results suggest that immediately after training there is an engagement of both pre- and postsynaptic voltage-sensitive calcium channels, followed by an increased reponse to N-methyl-D-aspartate receptor stimulation, and coinciding with the enhanced calcium-dependent glutamate release and an increase in N-methyl-D-aspartate-sensitive glutamate receptor binding that has been reported previously. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-sensitive mechanisms are activated at a later stage of memory formation, when increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate binding to glutamate receptors has been reported. Thus, responsiveness of calcium channels to agonist stimulation is implicated in temporally diverse stages in the cascade of events involved in memory formation following passive avoidance training in the chick.
Asunto(s)
Reacción de Prevención/fisiología , Química Encefálica/fisiología , Canales de Calcio/fisiología , Sinaptosomas/química , Sinaptosomas/fisiología , Animales , Calcio/metabolismo , Pollos , Estimulación Eléctrica , Agonistas de Aminoácidos Excitadores/farmacología , Conducta Alimentaria/fisiología , Femenino , Ligandos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/análisis , Memoria/fisiología , N-Metilaspartato/farmacología , Cloruro de Potasio/farmacología , Receptores de Glutamato/fisiología , Fracciones Subcelulares/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , ortoaminobenzoatosRESUMEN
In vivo microdialysis combined with measurements of 45Ca efflux from pre-labelled rat hippocampus has been utilised in our laboratory to demonstrate NMDA-evoked 45Ca2+ release to dialysate, reflecting calcium-induced calcium release (CICR) via ryanodine receptors (RyR). In the present study we attempted to reproduce this phenomenon in the rabbit hippocampus. Application of 1 mM NMDA to dialysis medium induced a decrease in Ca2+ concentration in dialysate, as a result of extracellular Ca2+ influx to neurones. The release of 45Ca2+ was not observed, instead a decrease in 45Ca2+ efflux rate from the NMDA treated rabbit hippocampus was noted, along with release to dialysate of prostaglandin D2, taurine and phosphoethanolamine. All these effects, reflecting different steps of intracellular calcium signalling, were insensitive to 100 microM dantrolene and 50 microM ryanodine, RyR modulators known to interfere with NMDA-evoked 45Ca2+ release in the rat hippocampus. Thus, although the results of this study demonstrate the role of extracellular Ca2+ influx to neurones in NMDA-evoked generation of Ca2+ signal in the rabbit hippocampus, the activity of CICR was not detected.
Asunto(s)
Calcio/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/metabolismo , N-Metilaspartato/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Etanolaminas/metabolismo , Microdiálisis , Prostaglandina D2/metabolismo , Conejos , Taurina/metabolismoRESUMEN
Excitotoxic hypothesis of ischemic brain lesion postulates a causal relation between stimulation of glutamatergic receptors and potentially neurotoxic neuronal calcium overload. In this short review we sum up our microdialysis studies on NMDA- and ischemia-evoked CA2+ fluxes in the rabbit hippocampus, which indicate that NMDA channels in normal conditions represent a potential route of Ca2+ influx to the hippocampal neurones, and in forebrain ischemia they significantly contribute to Ca2+ fluxes. These conclusions contrast with other data indicating that NMDA receptors play a marginal role in Ca2+ currents to neurones during advanced forebrain ischemia. A possible inhibition of NMDA receptors in ischemia by acidosis and nitric oxide has been postulated. Our recent data indicate that in brain microdialysis experiments local stabilisation of neutral brain parenchymal pH may prevent acidosis-induced inhibition of NMDA channels during ischemia, leading to overestimation of their role in calcium fluxes.
Asunto(s)
Isquemia Encefálica/metabolismo , Calcio/metabolismo , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , ConejosRESUMEN
In vivo microdialysis of the rabbit hippocampus was used to study the effects of N-methyl-D-aspartate (NMDA) receptor stimulation on dialysate concentrations of thromboxane B2 (Tx B2)- and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha)-immunoreactive materials that are stable metabolites of biologically active thromboxane A2 and prostacyclin. All pharmacological substances were applied in the dialysis medium. The application of 1 mM NMDA for 20 min resulted in five- and eightfold increases in Tx B2 and 6-keto PGF1 alpha concentrations, respectively. An increase in NMDA concentration to 2.5 mM did not potentiate a peak eicosanoid release, but significantly prolonged this effect. Either 10 microM MK-801 or the extrusion of Ca2+ from the dialysis medium inhibited the release by about 50%. Quinacrine, a phospholipase A2 inhibitor (250 microM), decreased the NMDA-evoked eicosanoid release by 30%, whereas 10 microM indomethacin, a cyclo-oxygenase inhibitor, completely suppressed the release. One hundred micromolar furegrelate, an inhibitor of thromboxane synthase, reduced by 75% Tx B2 release with concomitant 100% increase in 6-keto PGF1 alpha formation. Thus, stimulation of NMDA receptors induces calcium-dependent formation of thromboxane A2 and prostacyclin in the hippocampus, which may have pathophysiological implications. The neuronal site of their formation seems probable, although a transcellular mechanism of their synthesis should be also considered.
Asunto(s)
Hipocampo/metabolismo , N-Metilaspartato/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , 6-Cetoprostaglandina F1 alfa/biosíntesis , Animales , Calcio/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Microdiálisis , Conejos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Tromboxano B2/biosíntesisRESUMEN
Microdialysis was used to apply 1 mM N-methyl-D-aspartate (NMDA) for 20 min to the hippocampus of rabbits, control and pre-treated with GM1 ganglioside (im injections of 30 mg/kg for 3 d, twice a day). Concentrations of ionized Ca2+ and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha)-immunoreactive material in the dialyzates and 45Ca and [14C]sucrose efflux from the prelabeled hippocampus were determined. After 24 h, the morphology of the hippocampal neurons was examined. In control animals, the application of NMDA resulted in 25% decrease in Ca2+ concentration and in 1000% increase in 6-keto PGF 1 alpha concentration in the dialyzates. A 30% decrease in 45Ca efflux was accompanied by 20% increase in [14C]sucrose efflux, reflecting a corresponding reduction of the extracellular space volume. A degeneration of CA1 pyramidal neurons in the vicinity of a microdialysis probe was observed. In GM1-treated rabbits the NMDA-induced decrease in Ca2+ concentrations in the dialyzates was not reduced significantly, whereas a 70% stimulation of 45Ca efflux was noted, with a concomitant 40% reduction of 6-keto-PG F1 alpha release. NMDA-evoked increase in [14C]sucrose efflux did not differ from the control. In these animals CA1 neurons were well preserved. These results indicate that the pretreatment with GM1 results in activation of calcium extrusion from the NMDA-stimulated rabbit hippocampal neurons that alleviates destabilization of calcium homeostasis and reduces NMDA-evoked neuronal injury.
Asunto(s)
Calcio/metabolismo , Gangliósido G(M1)/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , N-Metilaspartato/antagonistas & inhibidores , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Radioisótopos de Calcio , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Microdiálisis , N-Metilaspartato/toxicidad , Conejos , Sacarosa/metabolismoRESUMEN
Using superfusion with albumin-containing medium of hippocampal and striatal slices of adult and developing rats at postnatal days (PND) 7-10, prelabelled with [3H]arachidonic acid ([3H]AA), we detected N-methyl-D-aspartate (NMDA) evoked release to the superfusion medium of radiolabelled material, 70% of which was associated with arachidonic acid (AA) and its metabolites. [3H]AA release was much more pronounced in PND 7-10 rats than in adults, and the response to NMDA in the hippocampal slices exceeded the reactions in the striatal slices. The subsequent experiments, employing only hippocampal slices of PND 7-10 rats, demonstrated that NMDA-stimulated [3H]AA release was dose-dependent in the micromolar range, was sensitive to NMDA receptor antagonists, and was inhibited in calcium-free medium and in the presence of quinacrine. [3H]AA release induced by 100 microM NMDA was not significantly inhibited by magnesium but was completely blocked by 7 Cl-kynurenic acid and ifenprodil (both antagonists 100 microM). The sulfhydryl reducing reagent dithiothreitol induced [3H]AA release; this response was sensitive to NMDA receptor antagonists. These data indicate that the NMDA induced, calcium triggered, and phospholipase A2 dependent AA release is highly pronounced in the developing rat hippocampus. NMDA receptors mediating AA release in the hippocampus of PND 7-10 rats are subject to glycine, polyamine and redox modulation, but they show low sensitivity to Mg2+ inhibition.
Asunto(s)
Ácido Araquidónico/metabolismo , Hipocampo/metabolismo , N-Metilaspartato/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Neostriado/efectos de los fármacos , Neostriado/metabolismo , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/efectos de los fármacosRESUMEN
Hippocampal slices of rats at postnatal day 7 were submitted to superfusion with Ca(2+)- and Mg(2+)-free, bicarbonate buffered ion balanced medium, and perfusate concentrations of eicosanoids: thromboxane B2 and 6-keto prostaglandin F1 alpha were determined by the radioimmunoassay. It was noted that the permanent presence of Ca2+ increased the basal eicosanoid level, and in these conditions modulation of eicosanoid production was lost, whereas temporary, a 20 min application of 1.3 mM Ca2+ did not influence significantly eicosanoid release. A 20 min application of the anoxic/aglycemic medium containing calcium did not change the content of eicosanoids in superfusates. A significant stimulation of the thromboxane B2 and 6-keto prostaglandin F1 alpha release was noted provided the application of the experimental medium was accompanied by a 10 min arrest of superfusion. This effect was inhibited by MK-801 and quinacrine, suggesting an involvement of NMDA receptors and phospholipase A2. We propose that a model of anoxic/aglycemic superfusion with a stop flow period allows retention of endogenous glutamate in the extracellular fluid, resembling a similar effect during in vivo ischemia, whereas during a continuous superfusion glutamate is immediately washed out. Consequently, an application of the anoxic/aglycemic medium accompanied by a temporary arrest of superfusion represents more adequate in vitro model of ischemia than a constant superfusion with this medium. In these conditions NMDA receptors mediate eicosanoid release.
Asunto(s)
Isquemia Encefálica/etiología , Eicosanoides/metabolismo , Hipocampo/química , Hipoxia/complicaciones , N-Metilaspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , 6-Cetoprostaglandina F1 alfa/análisis , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Ácido Araquidónico/metabolismo , Calcio/farmacología , Técnicas de Cultivo , Maleato de Dizocilpina/farmacología , Eicosanoides/análisis , Quinacrina/farmacología , Radioinmunoensayo , Ratas , Ratas Wistar , Tromboxano B2/análisis , Tromboxano B2/metabolismoRESUMEN
In this study Mongolian gerbils were submitted to a normothermic bilateral carotid ligation lasting 5 min. A noncompetitive antagonist of NMDA receptors, MK-801, 0.8 mg/kg, was injected i.p. 30 min before ischemia, or the ganglioside GM1, 30 mg/kg, was given i.p. for 3 days, twice a day. The morphology of the hippocampal CA1 neurones and the brain content of cyclooxygenase metabolites of arachidonic acid: prostaglandin 6-keto PGF1 alpha and thromboxane Tx B2 were studied. Untreated ischemia induced the accumulation in brain of the 6-keto PGF1 alpha and Tx B2 immunoreactive materials, and resulted in a lesion of 70% of CA1 neurones. In the MK-801- and GM1-pretreated groups the postischemic levels of Tx B2 were significantly decreased. However MK-801 and GM1 did not prevent damage to the CA1 neurones in gerbils normothermic after ischemia, whereas a partial neuroprotection was observed in hypothermic, MK-801 treated gerbils. The results of this study indicate that NMDA receptors may participate in the mechanism of postischemic release of eicosanoids in brain. They also confirm a potential modulatory role of gangliosides. These results are discussed in terms of the involvement of cyclooxygenase metabolites of arachidonic acid in the mechanism of a selective delayed neuronal damage to the hippocampus CA1 after ischemia.
Asunto(s)
Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Maleato de Dizocilpina/farmacología , Gangliósido G(M1)/farmacología , Hipocampo/patología , Prostaglandinas/metabolismo , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Femenino , Gerbillinae , Masculino , Tromboxano B2/metabolismoRESUMEN
Microdialysis was used to apply 10 microM nimodipine to the hippocampus of rabbits submitted to 15-min global cerebral ischemia. Analysis of dialysate allowed determination of changes in extracellular fluid concentrations of calcium (Ca2+e) and amino acids and in blood-brain barrier (BBB) permeability to fluorescein. General physiological parameters and EEG were recorded throughout the experiments, and morphological changes in the hippocampus 3 h following ischemia were evaluated. Ischemia caused rapid disappearance of EEG activity, a decrease of Ca2+e, and a release of neuroactive amino acids. Enhanced BBB permeability to fluorescein and early morphological changes in hippocampal pyramidal neurones were noted. Intrahippocampal nimodipine infusion only slightly reduced the decrease of Ca2+e and did not change amino acid release and BBB permeability after ischemia. However, nimodipine accelerated reappearance of hippocampal EEG activity and improved its pattern. A reduction of morphological changes of hippocampal pyramidal neurones in the vicinity of the dialysis probe was noted in the nimodipine-treated group. These results suggest that nimodipine-sensitive L channels do not play a critical role in ischemia-evoked Ca2+ redistribution in the rabbit hippocampus. It seems that a portion of the direct neuroprotective action of nimodipine may not be related to the inhibition of calcium influx into neurones.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Hipocampo/efectos de los fármacos , Nimodipina/administración & dosificación , Administración Tópica , Animales , Isquemia Encefálica/fisiopatología , Electrofisiología , Hipocampo/fisiopatología , Microdiálisis , Nimodipina/uso terapéutico , ConejosRESUMEN
Calcium ions are known to play a key role in the mechanism of excitotoxic and ischemic neuronal injury. Hippocampal CA1 neurons are selectively susceptible to this kind of damage. Although various calcium ionophores have been identified in the hippocampal neurons, it is not clear what the main pathway for Ca2+ entry is during overexcitation. These studies were aimed to estimate a potential contribution of different types of calcium ionophores in calcium redistribution to hippocampal neurons in vivo. The local microdialysis technique, combined with the 45Ca2+ utilizing method was used to measure the changes in extracellular Ca2+ concentrations ([Ca2+]e) in the rabbit hippocampus in vivo, and to apply active substances directly to the hippocampus. The application of N-methyl-D-aspartate (NMDA) resulted in a large, dose-dependent decrease of [Ca2+]e, which was sensitive to APV and MK-801, but was only slightly reduced by nimodipine and amiloride. The effect of high potassium medium was less pronounced and only slightly inhibited by nimodipine. However it was inhibited by 75% in the presence of MK-801 and then completely cancelled by nimodipine. To visualize the depolarization-induced calcium influx to hippocampal cells, KCl-induced cellular swelling and resulting shrinkage of the extracellular space, monitored with [U-14C]sucrose, was taken into account in calculating these data. These results indicate that calcium redistribution into hippocampal neurons through NMDA channels may highly exceed calcium fluxes in the hippocampus, attributable to a stimulation of the L-type voltage-sensitive calcium channels.
Asunto(s)
Calcio/fisiología , Glutamatos/toxicidad , Hipocampo/metabolismo , Animales , Calcio/metabolismo , Diálisis , Ácido Glutámico , Técnicas In Vitro , ConejosRESUMEN
This study evaluates the role of glycine in in vivo modulation of the activity of excitatory amino acid receptors sensitive to N-methyl-D-aspartate (NMDA) in the rabbit hippocampus. Changes of extracellular calcium concentration were studied by the microdialysis technique combined with 45Ca-utilizing clearance method. The steady state level of amino acids in the dialysate was analyzed with HPLC. Pharmacologically active substances were applied directly to the hippocampus via the dialysis medium. Glycine concentration in the extracellular fluid of the hippocampus was in the range of 10(-5) M. Application of NMDA induced a drop of calcium concentration in the extracellular compartment of the hippocampus. This effect was inhibited by 7-Cl-kynurenic acid, an antagonist of glycine binding site on the NMDA receptors. Application of glycine and D-serine to the dialysis medium did not interfere with the basal level of extracellular calcium in the hippocampus, but resulted in an enhancement of the NMDA-induced decrease of calcium concentration. These results indicate that in the hippocampus the NMDA receptors are under constant positive modulation by endogenous glycine. Since glycine binding sites on the NMDA receptors are not saturated by an endogenous ligand, glycine may play a role in regulation of the NMDA receptor activity in the hippocampus in vivo.
Asunto(s)
Calcio/farmacología , Glicina/farmacología , Hipocampo/metabolismo , N-Metilaspartato/farmacología , Aminoácidos/metabolismo , Animales , Calcio/metabolismo , Radioisótopos de Calcio , Diálisis , Sinergismo Farmacológico , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Hipocampo/efectos de los fármacos , Ácido Quinurénico/análogos & derivados , Ácido Quinurénico/farmacología , Conejos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Serina/farmacologíaRESUMEN
The N-methyl-D-aspartate (NMDA)-sensitive subtype of glutamate receptor, which gates Ca(2+)-permeable ion channels, is known for its role in learning and memory formation, in the induction of long-term potentiation, and also in seizure activity and neurotoxicity. In primary cultures of cerebellar neurons, agonists of NMDA receptors induce a dose-dependent release of [3H]arachidonic acid ([3H]AA), which is potentiated by activation of the glycine-positive modulatory site and inhibited by NMDA receptor antagonists. NMDA receptor-induced [3H]AA release is inhibited by quinacrine and partially depends on the presence of extracellular calcium. The [3H]AA release is not sensitive, however, to pretreatment with pertussis or cholera toxin, which suggests a Ca(2+)-dependent activation of phospholipase A2 not employing G proteins. Pretreatment of cultures with the natural and semisynthetic sphingolipids GT1b and PKS 3, respectively, inhibits NMDA receptor-mediated [3H]AA release. We also demonstrated glutamate-evoked [3H]AA release from rat hippocampal slices, which is NMDA receptor mediated, calcium dependent and sensitive to quinacrine. Arachidonic acid and its metabolites have been shown to play a role as second messengers and to modulate neuronal activity. Moreover, they are thought to act as transsynaptic modulators in the mechanism of NMDA receptor-induced long-term potentiation in the hippocampus. Their role in ischemic brain pathology has also been postulated. Our experiments on cultured cerebellar granule cells, incubated in a Mg(2+)-free medium deprived of glucose and oxygen, demonstrated a time-dependent stimulation of [3H]AA release. This release was inhibited by antagonists of NMDA receptors and by quinacrine. Stimulation of NMDA-sensitive glutamate receptors and the subsequent calcium-mediated activation of phospholipase A2 may play a role in the in vivo release of arachidonic acid during brain ischemia. This hypothesis is supported by the observation that the enhanced level of thromboxane B2 in the gerbil brain after 5 min of global ischemia is reduced by the systemic application of either the NMDA antagonist MK-801 or the ganglioside GM1.
Asunto(s)
Ácido Araquidónico/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Isquemia Encefálica/fisiopatología , Cerebelo/metabolismo , Gerbillinae , Hipocampo/metabolismo , Técnicas In Vitro , Neuronas/metabolismo , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Ratas , Transducción de Señal/fisiologíaRESUMEN
The effects of prostacyclin (PGI2) on ischemic changes of extracellular calcium concentration (CaE+2) and the blood-brain barrier (BBB) permeability were studied by microdialysis of the rabbit hippocampus. This was combined with morphological and neurophysiological observations. Complete cerebral ischemia lasting 15 min was produced by ligation of the brachiocephalic trunk, the left subclavian and both internal thoracic arteries. PGI2 was infused continuously i.v. in the last 3 min of ischemia and for 40 min after it, at a rate of 2 micrograms/kg/min. Control rabbits were submitted to untreated 15-min complete cerebral ischemia. The animals treated with PGI2 were found to have recovered bioelectric activity of the cortex and hippocampus in half the time that it took the untreated group. Application of PGI2 reduced by 60% the depth of ischemia-evoked drop of CaE+2 without acceleration of recovery during recirculation. The postischemic increase of BBB permeability to fluorescein was diminished. The number of morphologically changed neurons in the hippocampus of PGI2-treated animals was significantly lower than in the untreated group.